Protein-DNA selectivity is a central event in many biological processes, ranging from transcription and replication to restriction and modification. Type II restriction endonucleases are ideal systems for studying selectivity because of their high specificity and great variety. The enzymes recognize and cleave DNA sequences that vary between four to eight base pairs. Their sequence specificity is remarkable. A single base pair change within the recognition sequence can lead to well over a million fold reduction in activity. An understanding of sequence specific cleavage is relevant to protein mediating site-specific recombination and DNA repair by excision. The long-term goals of this project are to understand the mechanism by which type II restriction enzymes recognize and cleave DNA, and to design mutants with altered specificities. The three broad aims are: 1) Determine the basis of discrimination between closely related DNA sites. With structures of BamHI and Bglll in hand, we will now determine structures of an "intermediate" endonuclease with the specificity of both BamHI and Bglll: BstYI. We will also manipulate the specificity of BamHI with the aid of these new structures. 2) Determine the basis of specific versus non-specific DNA binding. We have determined the structure of BamHI bound to one non-cognate DNA sequence. We will now determine structures of BamHI bound to other non-cognate DNA sequences, in order to see the structural adaptations in going from one non-cognate sequence to another. We will also experimentally test a model of the non-cognate complex derived from theoretical analysis. 3) Determine the distinct mechanisms for targeting hydrolysis at a specific site. Endonucleases Fokl, Sill and Bsll recognize and cleave DNA by mechanisms that differ from most restriction enzymes. We will determine the structure of Bsll, which is unusual in its heterotetrameric architecture and has a clinical application in detecting cancerous mutations. We will complete the structure of Sill and cocrystallize the enzyme with a set of non-cognate and varied cognate DNA sites. Finally, we will complete our analysis of Fokl and cocrystallize it in an activated synaptic form with two DNA molecules.